Muscle Derived Cells for the Treatment of Urinary Tract Pathologies and Methods of Making and Using the Same

ABSTRACT

The present invention provides muscle-derived progenitor cells that show long-term survival following transplantation into body tissues and which can augment soft tissue following introduction (e.g. via injection, transplantation, or implantation) into a site of soft tissue. Also provided are methods of isolating muscle-derived progenitor cells, and methods of genetically modifying the cells for gene transfer therapy. The invention further provides methods of using compositions comprising muscle-derived progenitor cells for the augmentation and bulking of mammalian, including human, soft tissues in the treatment of various functional conditions, including malformation, injury, weakness, disease, or dysfunction. In particular, the present invention provides treatments and amelioration for urinary incontinence and other urinary tract pathologies.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.13/336,332, filed on Dec. 23, 2011, which is a divisional of U.S.application Ser. No. 12/013,076, now U.S. Pat. No. 8,105,834, filed onJan. 11, 2008, which claims benefit of priority from U.S. ProvisionalApplication No. 60/884,478, filed on Jan. 11, 2007, the contents ofwhich are incorporated herein in their entirety.

GOVERNMENT INTERESTS

This invention was made with Government support under Grant No. DK055387awarded by the National Institutes of Health. The Government has certainrights in this invention.

FIELD OF THE INVENTION

The present invention relates to muscle-derived progenitor cells (MDC)and compositions of MDCs and their use in the augmentation of bodytissues, particularly soft tissue like urethral and periurethral muscle.In particular, the present invention relates to muscle-derivedprogenitor cells that show long-term survival following introductioninto soft tissues, methods of isolating MDCs, and methods of usingMDC-containing compositions for the augmentation of human or animal softtissues, including epithelial, adipose, nerve, organ, muscle, ligament,and cartilage tissue. The invention also relates to novel uses ofmuscle-derived progenitor cells for the treatment of functionalconditions, such as stress urinary incontinence or urinary incontinence.

BACKGROUND OF THE INVENTION

Stress urinary incontinence (SUI) is a common condition and ischaracterized as the involuntary leakage of urine on effort, exertion,sneezing or coughing. (Abrams P, et al. Neurourol Urodyn 2002; 21(2):167-178). The etiology of SUI is multifactorial, involving damage and/orfunctional impairment of muscle and associated nerves that may occur asa result of advancing age, hormonal status, and pelvic floor damageresulting from vaginal child-birth. Due to the multifactorial etiology,a single treatment option, that is not limited in some fashion, does notcurrently exist.

The use of periurethral injectables as a minimally invasive treatmentoption, which may be performed on an outpatient basis under localanesthesia. This method of treatment is more cost effective in thenear-term, with shorter hospitalization, reduced operating room time,and generally fewer complications when compared with invasive surgicalapproaches such as bladder neck suspension. (Berman C J, et al. J Urol1997; 157(1): 122-124). However, it has disadvantages such as need formultiple injections due to loss of the long-term bulking effect owing todegradation, reabsorption, and/or migration, as well as otherimpediments such as bladder outlet obstruction and allergic reactions.Thus, there is a need for other, different urinary augmentationmaterials that are long-lasting, compatible with a wide range of hosttissues, and which cause minimal inflammation, scarring, and/orstiffening of the tissues surrounding the implant site.

Muscle derived cells isolated from rats have shown some success modelsfor urinary incontinence. (Cannon T W, et al. Urology 2003; 62(5):958-963 and Lee J Y et al. Int Urogynecol J Pelvic Floor Dysfunct 2003;14(1): 31-37; discussion 37). The instant invention provides the use ofhuman skeletal muscle derived cells (MDC) as an injectable treatment forSUI, and other urinary tract pathologies.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide novel humanmuscle-derived progenitor cells (MDCs) and human MDC compositionsexhibiting long-term survival following transplantation. The MDCs ofthis invention and compositions containing the MDCs comprise earlyprogenitor muscle cells, i.e., muscle-derived stem cells, that expressprogenitor cell markers, such as desmin, M-cadherin, MyoD, myogenin,CD34, and Bcl-2. In addition, these early progenitor muscle cellsexpress the Flk-1, Sca-1, MNF, and c-met cell markers, but do notexpress the CD45 or c-Kit cell markers.

It is another object of the present invention to provide methods forisolating and enriching human muscle-derived progenitor cells from astarting muscle cell population. These methods result in the enrichmentof human MDCs that have long-term survivability after transplantation orintroduction into a site of soft tissue. The MDC population according tothe present invention is particularly enriched with cells that expressprogenitor cell markers, such as desmin, M-cadherin, MyoD, myogenin,CD34, and Bcl-2. This MDC population also expresses the Flk-1, Sca-1,MNF, and c-met cell markers, but does not express the CD45 or c-Kit cellmarkers.

It is yet another object of the present invention to provide methods ofusing MDCs and compositions comprising MDCs for the augmentation ofmuscle soft tissue, or non-muscle soft tissue, including smooth muscle,and various organ tissues, without the need for polymer carriers orspecial culture media for transplantation. Such methods include theadministration of MDC compositions by introduction into soft tissue, forexample by direct injection into tissue, or by systemic distribution ofthe compositions. Preferably, soft tissue includes non-bone bodytissues. More preferably, soft tissue includes non-striated muscle andnon-bone body tissues. Most preferably, soft tissue includes non-muscle,non-bone body tissues. As used herein, augmentation refers to filling,bulking, supporting, enlarging, extending, or increasing the size ormass of body tissue.

It is another object of the present invention to provide methods ofaugmenting soft tissue, either muscle-derived soft tissue, ornon-muscle-derived soft tissue, following injury, wounding, surgeries,traumas, non-traumas, or other procedures that result in fissures,openings, depressions, wounds, and the like, in the skin or in internalsoft tissues or organs.

It is yet another object of the present invention to provide humanMDC-based treatments for urinary tract disease and associated symptoms.Pharmaceutical compositions comprising MDCs and compositions comprisingMDCs may be used for the treatment of urinary tract pathologies. Thesepharmaceutical compositions comprise isolated human MDCs. These MDCs maybe subsequently expanded by cell culture after isolation. In oneembodiment of the invention, these MDCs are frozen prior to delivery toa subject in need of the pharmaceutical composition.

In one embodiment, when the human MDCs and compositions thereof are usedto treat urinary incontinence they are injected directly into theurethra. Preferably, they may be injected into the periurethral muscleIn another embodiment, human MDCs and compositions thereof are used toimprove at least one symptom of urinary tract disease. These symptomsinclude urinary incontinence, urinary tract infection, frequenturination, painful urination, burning sensation when urinating, fatigue,tremor, cloudy urine, blood in urine, and kidney infection.

Human MDCs are isolated from a biopsy of skeletal muscle. In oneembodiment, the skeletal muscle from the biopsy may be stored for 1-6days. In one aspect of this embodiment, the skeletal muscle from thebiopsy is stored at 4° C. The MDCs are then isolated using the pre-plateor the single plate technique.

Using the pre-plate technique, a suspension of skeletal muscle cellsfrom skeletal muscle tissue is plated in a first container to whichfibroblast cells of the skeletal muscle cell suspension adhere.Non-adherent cells are then re-plated in a second container, wherein thestep of re-plating is after 15-20% of cells have adhered to the firstcontainer. This replating step must be repeated at least once. The MDCsare thereby isolated and may be administered to the esophagus of themammalian subject.

Using the single plate technique, the cells are minced, and digestedusing a collagenase, dispase, another enzyme or a combination ofenzymes. After washing the enzyme from the cells, the cells are culturedin a flask in culture medium for between about 30 and about 120 minutes.During this period of time, the “rapidly adhering cells” stick to thewalls of the flask or container, while the “slowly adhering cells” orMDCs remain in suspension. The “slowly adhering cells” are transferredto a second flask or container and cultured therein for a period of 1-3days. During this second period of time the “slowly adhering cells” orMDCs stick to the walls of the second flask or container.

In another embodiment of the invention, these MDCs are expanded to anynumber of cells. In a preferred aspect of this embodiment, the cells areexpanded in new culture media for between about 10 and 20 days. Morepreferably, the cells are expanded for 17 days.

The MDCs, whether expanded or not expanded, may be preserved in order tobe transported or stored for a period of time before use. In oneembodiment, the MDCs are frozen. Preferably, the MDCs are frozen atbetween about −20 and −90° C. More preferably, the MDCs are frozen atabout −80° C. These frozen MDCs are used as a pharmaceuticalcomposition.

Additional objects and advantages afforded by the present invention willbe apparent from the detailed description and exemplificationhereinbelow.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or patent application file contains at least one photographicreproduction executed in color. Copies of this patent or patentapplication with color photographic reproduction(s) will be provided bythe U.S. Patent and Trademark Office upon request and payment of thenecessary fee.

The appended drawings of the figures are presented to further describethe invention and to assist in its understanding through clarificationof its various aspects.

FIG. 1 is a bar graph showing higher leak point pressure in rats treatedwith human MDCs than control.

FIG. 2A is a light micrograph of a proximal urethral sphincter in acontrol rat.

FIG. 2B is a light micrograph of a proximal urethral sphincter in a rattreated with human MDCs.

FIG. 3 shows immunofluorescent labeling with a human-specific nuclearantibody (lamins A/C) revealing the presence of human nucleiincorporated within the striated sphincter muscle layer in human MDCinjected tissue (100×). Arrows point to individual nuclei.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides human MDCs and methods of using such cells togenerate tissue with bulking properties that have the potential toimprove coaptation and intrinsic sphincter function by remodeling thedamaged tissue. The invention further provides methods of treatingurinary tract disorders including incontinence and stress urinaryincontinence. The isolation of human muscle-derived cells (MDCs) fromadult tissue are capable of achieving functional success within anestablished urethral sphincter injury model.

Muscle-Derived Cells and Compositions

The present invention provides MDCs comprised of early progenitor cells(also termed muscle-derived progenitor cells or muscle-derived stemcells herein) that show long-term survival rates followingtransplantation into body tissues, preferably soft tissues. To obtainthe MDCs of this invention, a muscle explant, preferably skeletalmuscle, is obtained from an animal donor, preferably from a mammal,including rats, dogs and humans. This explant serves as a structural andfunctional syncytium including “rests” of muscle precursor cells (T. A.Partridge et al., 1978, Nature 73:306-8; B. H. Lipton et al., 1979,Science 205:12924).

Cells isolated from primary muscle tissue contain mixture offibroblasts, myoblasts, adipocytes, hematopoietic, and muscle-derivedprogenitor cells. The progenitor cells of a muscle-derived populationcan be enriched using differential adherence characteristics of primarymuscle cells on collagen coated tissue flasks, such as described in U.S.Pat. No. 6,866,842 of Chancellor et al. Cells that are slow to adheretend to be morphologically round, express high levels of desmin, andhave the ability to fuse and differentiate into multinucleated myotubes(U.S. Pat. No. 6,866,842 of Chancellor et al.). A subpopulation of thesecells was shown to respond to recombinant human bone morphogenic protein2 (rhBMP-2) In vitro by expressing increased levels of alkalinephosphatase, parathyroid hormone dependent 3′,5′-cAMP, and osteogeniclineage and myogenic lineages (U.S. Pat. No. 6,866,842 of Chancellor etal.; T. Katagiri et al., 1994, J. Cell Biol., 127:1755-1766).

In one embodiment of the invention, a preplating procedure may be usedto differentiate rapidly adhering cells from slowly adhering cells(MDCs). In accordance with the present invention, populations of rapidlyadhering cells (PP1-4) and slowly adhering, round MDCs (PP6) wereisolated and enriched from skeletal muscle explants and tested for theexpression of various markers using immunohistochemistry to determinethe presence of pluripotent cells among the slowly adhering cells(Example 1; patent application U.S. Ser. No. 09/302,896 of Chancellor etal.). The PP6 cells expressed myogenic markers, including desmin, MyoD,and Myogenin. The PP6 cells also expressed c-met and MNF, two geneswhich are expressed at an early stage of myogenesis (J. B. Miller etal., 1999, Curr. Top. Dev. Biol. 43:191-219). The PP6 showed a lowerpercentage of cells expressing M-cadherin, a satellite cell-specificmarker (A. Irintchev et al., 1994, Development Dynamics 199:326-337),but a higher percentage of cells expressing Bcl-2, a marker limited tocells in the early stages of myogenesis (J. A. Dominov et al., 1998, J.Cell Biol. 142:537-544). The PP6 cells also expressed CD34, a markeridentified with human hematopoietic progenitor cells, as well as stromalcell precursors in bone marrow (R. G. Andrews et al., 1986, Blood67:842-845; C. I. Civin et al., 1984, J. Immunol. 133:157-165; L. Finaet al, 1990, Blood 75:2417-2426; P. J. Simmons et al., 1991, Blood78:2848-2853). The PP6 cells also expressed Flk-1, a mouse homologue ofhuman KDR gene which was recently identified as a marker ofhematopoietic cells with stem cell-like characteristics (B. L. Ziegleret al., 1999, Science 285:1553-1558). Similarly, the PP6 cells expressedSca-1, a marker present in hematopoietic cells with stem cell-likecharacteristics (M. van de Rijn et al., 1989, Proc. Natl. Acad. Sci. USA86:4634-8; M. Osawa et al., 1996, J. Immunol. 156:3207-14). However, thePP6 cells did not express the CD45 or c-Kit hematopoietic stem cellmarkers (reviewed in L K. Ashman, 1999, Int. J. Biochem. Cell. Biol.31:1037-51; G. A. Koretzky, 1993, FASEB J. 7:420-426).

One embodiment of the present invention is the PP6 population ofmuscle-derived progenitor cells having the characteristics describedherein. These muscle-derived progenitor cells express the desmin, CD34,and Bcl-2 cell markers. In accordance with the present invention, thePP6 cells are isolated by the techniques described herein (Example 1) toobtain a population of muscle-derived progenitor cells that havelong-term survivability following transplantation. The PP6muscle-derived progenitor cell population comprises a significantpercentage of cells that express progenitor cell markers such as desmin,CD34, and Bcl-2. In addition, PP6 cells express the Flk-1 and Sca-1 cellmarkers, but do not express the CD45 or c-Kit markers. Preferably,greater than 95% of the PP6 cells express the desmin, Sca-1, and Flk-1markers, but do not express the CD45 or c-Kit markers. It is preferredthat the PP6 cells are utilized within about 1 day or about 24 hoursafter the last plating.

In a preferred embodiment, the rapidly adhering cells and slowlyadhering cells (MDCs) are separated from each other using a singleplating technique. One such technique is described in Example 2. First,cells are provided from a skeletal muscle biopsy. The biopsy need onlycontain about 100 mg of cells. Biopsies ranging in size from about 50 mgto about 500 mg can be used according to both the pre-plating and singleplating methods of the invention. Further biopsies of 50, 100, 110, 120,130, 140, 150, 200, 250, 300, 400 and 500 mg can be used according toboth the pre-plating and single plating methods of the invention.

In a preferred embodiment of the invention, the tissue from the biopsyis then stored for 1 to 7 days. This storage is at a temperature fromabout room temperature to about 4° C. This waiting period causes thebiopsied skeletal muscle tissue to undergo stress. While this stress isnot necessary for the isolation of MDCs using this single platetechnique, it seems that using the wait period results in a greateryield of MDCs.

Tissue from the biopsies is minced and centrifuged. The pellet isresuspended and digested using a digestion enzyme. Enzymes that may beused include collagenase, dispase or combinations of these enzymes.After digestion, the enzyme is washed off of the cells. The cells aretransferred to a flask in culture media for the isolation of the rapidlyadhering cells. Many culture media may be used. Particularly preferredculture media include those that are designed for culture of endothelialcells including Cambrex Endothelial Growth Medium. This medium may besupplemented with other components including fetal bovine serum, IGF-1,bFGF, VEGF, EGF, hydrocortisone, heparin, and/or ascorbic acid. Othermedia that may be used in the single plating technique include InCellM310F medium. This medium may be supplemented as described above, orused unsupplemented.

The step for isolation of the rapidly adhering cells may require culturein flask for a period of time from about 30 to about 120 minutes. Therapidly adhering cells adhere to the flask in 30, 40, 50, 60, 70, 80,90, 100, 110 or 120 minutes. After they adhere, the slowly adheringcells are separated from the rapidly adhering cells by removing theculture media from the flask to which the rapidly adhering cells areattached.

The culture medium removed from this flask is then transferred to asecond flask. The cells may be centrifuged and resuspended in culturemedium before being transferred to the second flask. The cells arecultured in this second flask for between 1 and 3 days. Preferably, thecells are cultured for two days. During this period of time, the slowlyadhering cells (MDCs) adhere to the flask. After the MDCs have adhered,the culture media is removed and new culture media is added so that theMDCs can be expanded in number. The MDCs may be expanded in number byculturing them for from about 10 to about 20 days. The MDCs may beexpanded in number by culturing them for 10, 11, 12, 13, 14, 15, 16, 17,18, 19 or 20 days. Preferably, the MDCs are subject to expansion culturefor 17 days.

As an alternative to the pre-plating and single plating methods, theMDCs of the present invention can be isolated by fluorescence-activatedcell sorting (FACS) analysis using labeled antibodies against one ormore of the cell surface markers expressed by the MDCs (C. Webster etal., 1988, Exp. Cell. Res. 174:252-65; J. R. Blanton et al., 1999,Muscle Nerve 22:43-50). For example, FACS analysis can be performedusing labeled antibodies to directed to CD34, Flk-1, Sca-1, and/or theother cell-surface markers described herein to select a population ofPP6-like cells that exhibit long-term survivability when introduced intothe host tissue. Also encompassed by the present invention is the use ofone or more fluorescence-detection labels, for example, fluorescein orrhodamine, for antibody detection of different cell marker proteins.

Using any of the MDCs isolation methods described above, MDCs that areto be transported, or are not going to be used for a period of time maybe preserved using methods known in the art. More specifically, theisolated MDCs may be frozen to a temperature ranging from about −25 toabout −90° C. Preferably, the MDCs are frozen at about −80° C., on dryice for delayed use or transport. The freezing may be done with anycryopreservation medium known in the art.

Muscle-Derived Cell-Based Treatments

In one embodiment of the present invention, the MDCs are isolated from askeletal muscle source and introduced or transplanted into a muscle ornon-muscle soft tissue site of interest, or into bone structures.Advantageously, the MDCs of the present invention are isolated andenriched to contain a large number of progenitor cells showing long-termsurvival following transplantation. In addition, the muscle-derivedprogenitor cells of this invention express a number of characteristiccell markers, such desmin, CD34, and Bcl-2. Furthermore, themuscle-derived progenitor cells of this invention express the Sca-1, andFlk-1 cell markers, but do not express the CD45 or c-Kit cell markers.

MDCs and compositions comprising MDCs of the present invention can beused to repair, treat, or ameliorate various aesthetic or functionalconditions (e.g. defects) through the augmentation of muscle ornon-muscle soft tissues. In particular, such compositions can be used assoft-tissue bulking agents for the treatment of urinary incontinence andother instances of smooth muscle weakness, disease, injury, ordysfunction. In addition, such MDCs and compositions thereof can be usedfor augmenting soft tissue not associated with injury by adding bulk toa soft tissue area, opening, depression, or void in the absence ofdisease or trauma, such as for “smoothing”. Multiple and successiveadministrations of MDCs are also embraced by the present invention.

For MDC-based treatments, a skeletal muscle explant is preferablyobtained from an autologous or heterologous human or animal source. Anautologous animal or human source is more preferred. MDC compositionsare then prepared and isolated as described herein. To introduce ortransplant the MDCs and/or compositions comprising the MDCs according tothe present invention into a human or animal recipient, a suspension ofmononucleated muscle cells is prepared. Such suspensions containconcentrations of the muscle-derived progenitor cells of the inventionin a physiologically-acceptable carrier, excipient, or diluent. Forexample, suspensions of MDCs for administering to a subject can comprise10⁸ to 10⁹ cells/ml in a sterile solution of complete medium modified tocontain the subject's serum, as an alternative to fetal bovine serum.Alternatively, MDC suspensions can be in serum-free, sterile solutions,such as cryopreservation solutions (Celox Laboratories, St. Paul,Minn.). The MDC suspensions can then be introduced e.g., via injection,into one or more sites of the donor tissue.

The described cells can be administered as a pharmaceutically orphysiologically acceptable preparation or composition containing aphysiologically acceptable carrier, excipient, or diluent, andadministered to the tissues of the recipient organism of interest,including humans and non-human animals. The MDC-containing compositioncan be prepared by resuspending the cells in a suitable liquid orsolution such as sterile physiological saline or other physiologicallyacceptable injectable aqueous liquids. The amounts of the components tobe used in such compositions can be routinely determined by those havingskill in the art.

The MDCs or compositions thereof can be administered by placement of theMDC suspensions onto absorbent or adherent material, i.e., a collagensponge matrix, and insertion of the MDC-containing material into or ontothe site of interest. Alternatively, the MDCs can be administered byparenteral routes of injection, including subcutaneous, intravenous,intramuscular, and intrasternal. Other modes of administration include,but are not limited to, intranasal, intrathecal, intracutaneous,percutaneous, enteral, and sublingual. In one embodiment of the presentinvention, administration of the MDCs can be mediated by endoscopicsurgery.

For injectable administration, the composition is in sterile solution orsuspension or can be resuspended in pharmaceutically- andphysiologically-acceptable aqueous or oleaginous vehicles, which maycontain preservatives, stabilizers, and material for rendering thesolution or suspension isotonic with body fluids (i.e. blood) of therecipient. Non-limiting examples of excipients suitable for use includewater, phosphate buffered saline, pH 7.4, 0.15 M aqueous sodium chloridesolution, dextrose, glycerol, dilute ethanol, and the like, and mixturesthereof. Illustrative stabilizers are polyethylene glycol, proteins,saccharides, amino acids, inorganic acids, and organic acids, which maybe used either on their own or as admixtures. The amounts or quantities,as well as the routes of administration used, are determined on anindividual basis, and correspond to the amounts used in similar types ofapplications or indications known to those of skill in the art.

To optimize transplant success, the closest possible immunological matchbetween donor and recipient is desired. If an autologous source is notavailable, donor and recipient Class I and Class II histocompatibilityantigens can be analyzed to determine the closest match available. Thisminimizes or eliminates immune rejection and reduces the need forimmunosuppressive or immunomodulatory therapy. If required,immunosuppressive or immunomodulatory therapy can be started before,during, and/or after the transplant procedure. For example, cyclosporinA or other immunosuppressive drugs can be administered to the transplantrecipient. Immunological tolerance may also be induced prior totransplantation by alternative methods known in the art (D. J. Watt etal., 1984, Clin. Exp. Immunol. 55:419; D. Faustman et al., 1991, Science252:1701).

Consistent with the present invention, the MDCs can be administered tobody tissues, including epithelial tissue (i.e., skin, lumen, etc.)muscle tissue (i.e. smooth muscle), and various organ tissues such asthose organs that are associated with the urological system (i.e.,bladder, urethra, ureter, kidneys, etc.).

The number of cells in an MDC suspension and the mode of administrationmay vary depending on the site and condition being treated. Asnon-limiting examples, in accordance with the present invention, about3-5×10⁵ MDCs are injected for the treatment of urinary incontinence (seeExample 3). Consistent with the Examples disclosed herein, a skilledpractitioner can modulate the amounts and methods of MDC-basedtreatments according to requirements, limitations, and/or optimizationsdetermined for each case.

Conditions of the lumen: In another embodiment, the MDCs andcompositions thereof according to the present invention have furtherutility as treatments for conditions of the lumen in an animal or mammalsubject, including humans. Specifically, the muscle-derived progenitorcells are used for completely or partially blocking, enhancing,enlarging, sealing, repairing, bulking, or filling various biologicallumens or voids within the body. Lumens include, without limitation theurethra. Voids may include, without limitation, various tissue wounds(i.e., loss of muscle and soft tissue bulk due to trauma; destruction ofsoft tissue due to penetrating projectiles such as a stab wound orbullet wound; loss of soft tissue from disease or tissue death due tosurgical removal of the tissue including loss of breast tissue followinga mastectomy for breast cancer or loss of muscle tissue followingsurgery to treat sarcoma, etc.), lesions, fissures, diverticulae, cysts,fistulae, aneurysms, and other undesirable or unwanted depressions oropenings that may exist within the body of an animal or mammal,including humans. For the treatment of conditions of the lumen, the MDCsare prepared as disclosed herein and then administered, e.g. viainjection or intravenous delivery, to the lumenal tissue to fill orrepair the void. The number of MDCs introduced is modulated to repairlarge or small voids in a soft tissue environment, as required.

Conditions of the sphincter: The MDCs and compositions thereof accordingto the present invention can also be used for the treatment of asphincter injury, weakness, disease, or dysfunction in an animal ormammal, including humans. In particular, the MDCs are used to augmenttissues of the urinary sphincters. More specifically, the presentinvention provides soft tissue augmentation treatments for urinaryincontinence. For the treatment of sphincter defects, the MDCs areprepared as described herein and then administered to the sphinctertissue, e.g. via injection, to provide additional bulk, filler, orsupport. The number of MDCs introduced is modulated to provide varyingamounts of bulking material as required. For example, about 3-5×10⁵ MDCsare injected for the treatment of urinary incontinence (see Example 3).

In addition, the MDCs and compositions thereof can be used to affectcontractility in smooth muscle tissue, such as urinary or bladdertissue, as example. Thus, the present invention also embraces the use ofMDCs of the invention in restoring muscle contraction, and/orameliorating or overcoming smooth muscle contractility problems.

Genetically Engineered Muscle-Derived Cells

In another aspect of the present invention, the MDCs of this inventionmay be genetically engineered to contain a nucleic acid sequence(s)encoding one or more active biomolecules, and to express thesebiomolecules, including proteins, polypeptides, peptides, hormones,metabolites, drugs, enzymes, and the like. Such MDCs may behistocompatible (autologous) or nonhistocompatible (allogeneic) to therecipient, including humans. These cells can serve as long-term localdelivery systems for a variety of treatments, for example, urinaryincontinence.

Preferred in the present invention are autologous muscle-derivedprogenitor cells, which will not be recognized as foreign to therecipient. In this regard, the MDCs used for cell-mediated gene transferor delivery will desirably be matched vis-a-vis the majorhistocompatibility locus (MHC or HLA in humans). Such MHC or HLA matchedcells may be autologous. Alternatively, the cells may be from a personhaving the same or a similar MHC or HLA antigen profile. The patient mayalso be tolerized to the allogeneic MHC antigens. The present inventionalso encompasses the use of cells lacking MHC Class I and/or IIantigens, such as described in U.S. Pat. No. 5,538,722.

The MDCs may be genetically engineered by a variety of moleculartechniques and methods known to those having skill in the art, forexample, transfection, infection, or transduction. Transduction as usedherein commonly refers to cells that have been genetically engineered tocontain a foreign or heterologous gene via the introduction of a viralor non-viral vector into the cells. Transfection more commonly refers tocells that have been genetically engineered to contain a foreign geneharbored in a plasmid, or non-viral vector. MDCs can be transfected ortransduced by different vectors and thus can serve as gene deliveryvehicles to transfer the expressed products into muscle.

Although viral vectors are preferred, those having skill in the art willappreciate that the genetic engineering of cells to contain nucleic acidsequences encoding desired proteins or polypeptides, cytokines, and thelike, may be carried out by methods known in the art, for example, asdescribed in U.S. Pat. No. 5,538,722, including fusion, transfection,lipofection mediated by the use of liposomes, electroporation,precipitation with DEAE-Dextran or calcium phosphate, particlebombardment (biolistics) with nucleic acid-coated particles (e.g., goldparticles), microinjection, and the like.

Vectors for introducing heterologous (i.e., foreign) nucleic acid (DNAor RNA) into muscle cells for the expression of bioactive products arewell known in the art. Such vectors possess a promoter sequence,preferably, a promoter that is cell-specific and placed upstream of thesequence to be expressed. The vectors may also contain, optionally, oneor more expressible marker genes for expression as an indication ofsuccessful transfection and expression of the nucleic acid sequencescontained in the vector.

Illustrative examples of vehicles or vector constructs for transfectionor infection of the muscle-derived cells of the present inventioninclude replication-defective viral vectors, DNA virus or RNA virus(retrovirus) vectors, such as adenovirus, herpes simplex virus andadeno-associated viral vectors. Adeno-associated virus vectors aresingle stranded and allow the efficient delivery of multiple copies ofnucleic acid to the cell's nucleus. Preferred are adenovirus vectors.The vectors will normally be substantially free of any prokaryotic DNAand may comprise a number of different functional nucleic acidsequences. Examples of such functional sequences include polynucleotide,e.g., DNA or RNA, sequences comprising transcriptional and translationalinitiation and termination regulatory sequences, including promoters(e.g., strong promoters, inducible promoters, and the like) andenhancers which are active in muscle cells.

Also included as part of the functional sequences is an open readingframe (polynucleotide sequence) encoding a protein of interest; flankingsequences may also be included for site-directed integration. In somesituations, the 5′-flanking sequence will allow homologousrecombination, thus changing the nature of the transcriptionalinitiation region, so as to provide for inducible or noninducibletranscription to increase or decrease the level of transcription, as anexample.

In general, the nucleic acid sequence desired to be expressed by themuscle-derived progenitor cell is that of a structural gene, or afunctional fragment, segment or portion of the gene, that isheterologous to the muscle-derived progenitor cell and encodes a desiredprotein or polypeptide product, for example. The encoded and expressedproduct may be intracellular, i.e., retained in the cytoplasm, nucleus,or an organelle of a cell, or may be secreted by the cell. Forsecretion, the natural signal sequence present in the structural genemay be retained, or a signal sequence that is not naturally present inthe structural gene may be used. When the polypeptide or peptide is afragment of a protein that is larger, a signal sequence may be providedso that, upon secretion and processing at the processing site, thedesired protein will have the natural sequence. Examples of genes ofinterest for use in accordance with the present invention include genesencoding cell growth factors, cell differentiation factors, cellsignaling factors and programmed cell death factors. Specific examplesinclude, but are not limited to, genes encoding BMP-2 (rhBMP-2), IL-1Ra,Factor IX, and connexin 43.

As mentioned above, a marker may be present for selection of cellscontaining the vector construct. The marker may be an inducible ornon-inducible gene and will generally allow for positive selection underinduction, or without induction, respectively. Examples of commonly-usedmarker genes include neomycin, dihydrofolate reductase, glutaminesynthetase, and the like.

The vector employed will generally also include an origin of replicationand other genes that are necessary for replication in the host cells, asroutinely employed by those having skill in the art. As an example, thereplication system comprising the origin of replication and any proteinsassociated with replication encoded by a particular virus may beincluded as part of the construct. The replication system must beselected so that the genes encoding products necessary for replicationdo not ultimately transform the muscle-derived cells. Such replicationsystems are represented by replication-defective adenovirus constructedas described, for example, by G. Acsadi et al., 1994, Hum. Mol. Genet3:579-584, and by Epstein-Barr virus. Examples of replication defectivevectors, particularly, retroviral vectors that are replicationdefective, are BAG, described by Price et al., 1987, Proc. Natl. Acad.Sci. USA, 84:156; and Sanes et al., 1986, EMBO J., 5:3133. It will beunderstood that the final gene construct may contain one or more genesof interest, for example, a gene encoding a bioactive metabolicmolecule. In addition, cDNA, synthetically produced DNA or chromosomalDNA may be employed utilizing methods and protocols known and practicedby those having skill in the art.

If desired, infectious replication-defective viral vectors may be usedto genetically engineer the cells prior to In vivo injection of thecells. In this regard, the vectors may be introduced into retroviralproducer cells for amphotrophic packaging. The natural expansion ofmuscle-derived progenitor cells into adjacent regions obviates a largenumber of injections into or at the site(s) of interest.

In another aspect, the present invention provides ex vivo gene deliveryto cells and tissues of a recipient mammalian host, including humans,through the use of MDCs, e.g., early progenitor muscle cells, that havebeen virally transduced using an adenoviral vector engineered to containa heterologous gene encoding a desired gene product. Such an ex vivoapproach provides the advantage of efficient viral gene transfer, whichis superior to direct gene transfer approaches. The ex vivo procedureinvolves the use of the muscle-derived progenitor cells from isolatedcells of muscle tissue. The muscle biopsy that will serve as the sourceof muscle-derived progenitor cells can be obtained from an injury siteor from another area that may be more easily obtainable from theclinical surgeon.

It will be appreciated that in accordance with the present invention,clonal isolates can be derived from the population of muscle-derivedprogenitor cells (i.e., PP6 cells or slowly adhering cells) usingvarious procedures known in the art, for example, limiting dilutionplating in tissue culture medium. Clonal isolates comprise geneticallyidentical cells that originate from a single, solitary cell. Inaddition, clonal isolates can be derived using FACS analysis asdescribed above, followed by limiting dilution to achieve a single cellper well to establish a clonally isolated cell line.

The MDCs are first infected with engineered viral vectors containing atleast one heterologous gene encoding a desired gene product, suspendedin a physiologically acceptable carrier or excipient, such as saline orphosphate buffered saline, and then administered to an appropriate sitein the host. Consistent with the present invention, the MDCs can beadministered to body tissues, including bone, epithelial tissue,connective tissue, muscle tissue, and various organ tissues such asthose organs that are associated with the digestive system,cardiovascular system, respiratory system, reproductive system,urological system, and nervous system, as described above. The desiredgene product is expressed by the injected cells, which thus introducethe gene product into the host. The introduced and expressed geneproducts can thereby be utilized to treat, repair, or ameliorate theinjury, dysfunction, or disease, due to their being expressed over longtime periods by the MDCs of the invention, having long-term survival inthe host.

In animal model studies of myoblast-mediated gene therapy, implantationof 10⁶ myoblasts per 100 mg muscle was required for partial correctionof muscle enzyme defects (see, J. E. Morgan et al., 1988, J. Neural.Sci. 86:137; T. A. Partridge et al., 1989, Nature 337:176).Extrapolating from this data, approximately 10¹² MDCs suspended in aphysiologically compatible medium can be implanted into muscle tissuefor gene therapy for a 70 kg human. This number of MDCs of the inventioncan be produced from a single 100 mg skeletal muscle biopsy from a humansource (see below). For the treatment of a specific injury site, aninjection of genetically engineered MDCs into a given tissue or site ofinjury comprises a therapeutically effective amount of cells in solutionor suspension, preferably, about 10⁵ to 10⁶ cells per cm³ of tissue tobe treated, in a physiologically acceptable medium.

Examples Example 1 MDC Enrichment, Isolation and Analysis According tothe Pre-Plating Method

Enrichment and isolation of MDCs: MDCs were prepared as described (U.S.Pat. No. 6,866,842 of Chancellor et al.). Muscle explants were obtainedfrom the hind limbs of a number of sources, namely from 1-week-old mdx(dystrophic) mice (C57BL/10ScSn mdx/mdx, Jackson Laboratories), 4-6week-old normal female SD (Sprague Dawley) rats, or SCID (severecombined immunodeficiency) mice. The muscle tissue from each of theanimal sources was dissected to remove any bones and minced into aslurry. The slurry was then digested by 1 hour serial incubations with0.2% type XI collagenase, dispase (grade II, 240 unit), and 0.1% trypsinat 37° C. The resulting cell suspension was passed through 18, 20, and22 gauge needles and centrifuged at 3000 rpm for 5 minutes.Subsequently, cells were suspended in growth medium (DMEM supplementedwith 10% fetal bovine serum, 10% horse serum, 0.5% chick embryo extract,and 2% penicillin/streptomycin). Cells were then preplated incollagen-coated flasks (U.S. Pat. No. 6,866,842 of Chancellor et al.).After approximately 1 hour, the supernatant was removed from the flaskand re-plated into a fresh collagen-coated flask. The cells whichadhered rapidly within this 1 hour incubation were mostly fibroblasts(Z. Qu et al., supra; U.S. Pat. No. 6,866,842 of Chancellor et al.). Thesupernatant was removed and re-plated after 30-40% of the cells hadadhered to each flask. After approximately 5-6 serial platings, theculture was enriched with small, round cells, designated as PP6 cells,which were isolated from the starting cell population and used infurther studies. The adherent cells isolated in the early platings werepooled together and designated as PP1-4 cells.

The mdx PP1-4, mdx PP6, normal PP6, and fibroblast cell populations wereexamined by immunohistochemical analysis for the expression of cellmarkers. The results of this analysis are shown in Table 1.

TABLE 1 Cell markers expressed in PP1-4 and PP6 cell populations. mdxPP1-4 mdx PP6 nor PP6 cells cells cells fibroblasts desmin +/− + + −CD34 − + + − Bcl-2 (−) + + − Flk-1 na + + − Sca-1 na + + − M-cadherin−/+ −/+ −/+ − MyoD −/+ +/− +/− − myogenin −/+ +/− +/− − Mdx PP1-4, mdxPP6, normal PP6, and fibroblast cells were derived by preplatingtechnique and examined by immunohistochemical analysis. “−” indicatesless than 2% of the cells showed expression; “(−)”; “−/+” indicates5-50% of the cells showed expression; “+/−” indicates ~40-80% of thecells showed expession; “+” indicates that >95% of the cells showedexpression; “nor” indicates normal cells; “na” indicates that theimmunohistochemical data is not available.

It is noted that both mdx and normal mice showed identical distributionof all of the cell markers tested in this assay. Thus, the presence ofthe mdx mutation does not affect the cell marker expression of theisolated PP6 muscle-cell derived population.

MDCs were grown in proliferation medium containing DMEM (Dulbecco'sModified Eagle Medium) with 10% FBS (fetal bovine serum), 10% HS (horseserum), 0.5% chick embryo extract, and 1% penicillin/streptomycin, orfusion medium containing DMEM supplemented with 2% fetal bovine serumand 1% antibiotic solution. All media supplies were purchased throughGibco Laboratories (Grand Island, N.Y.).

Example 2 MDC Enrichment, Isolation and Analysis According to the SinglePlate Method

Enrichment and Isolation of MDCs

Populations of rapidly- and slowly-adhering MDCs were isolated fromskeletal muscle of a mammalian subject. The subject may be a human, rat,dog or other mammal. Biopsy size ranged from 42 to 247 mg.

Skeletal muscle biopsy tissue was immediately placed in cold hypothermicmedium (HYPOTHERMOSOL® (BioLife) supplemented with gentamicin sulfate(100 ng/ml, Roche)) and stored at 4° C. After 3 to 7 days, biopsy tissuewas removed from storage and production was initiated. Any connective ornon-muscle tissue was dissected from the biopsy sample. The remainingmuscle tissue that was used for isolation is weighed. The tissue wasminced in Hank's Balanced Salt Solution (HBSS), transferred to a conicaltube, and centrifuged (2,500×g, 5 minutes). The pellet was thenresuspended in a Digestion Enzyme solution (Liberase Blendzyme 4(0.4-1.0 U/mL, Roche)). 2 mL of Digestion Enzyme solution was used per100 mg of biopsy tissue and was incubated for 30 minutes at 37° C. on arotating plate. The sample was then centrifuged (2,500×g, 5 minutes).The pellet was resuspended in culture medium and passed through a 70 μmcell strainer. The culture media used for the procedures described inthis Example was Cambrex Endothelial Growth Medium EGM-2 basal mediumsupplemented with the following components: i. 10% (v/v) fetal bovineserum, and ii. Cambrex EGM-2 SingleQuot Kit, which contains: InsulinGrowth Factor-1 (IGF-1), Basic Fibroblast Growth Factor (bFGF), VascularEndothelial Growth Factor (VEGF), Epidermal Growth Factor (EGF),Hydrocortisone, Heparin, and Ascorbic Acid. The filtered cell solutionwas then transferred to a T25 culture flask and incubated for 30-120minutes at 37° C. in 5% CO₂. Cells that attached to this flask weretermed the “rapidly-adhering cells”.

After incubation, the cell culture supernatant was removed from the T25flask and placed into a 15 mL conical tube. The T25 culture flask isrinsed with 2 mL of warmed culture medium and transferred to theaforementioned 15 mL conical tube. The 15 mL conical tube is centrifuged(2,500×g, 5 minutes). The pellet was resuspended in culture medium andtransferred to a new T25 culture flask. The flask was incubated for ˜2days at 37° C. in 5% CO2 (cells that attach to this flask were termedthe “slowly-adhering cells”). After incubation, the cell culturesupernatant was aspirated and new culture medium was added to the flask.The flask was then returned to the incubator for expansion. Standardculture passaging is carried out from here on to maintain the cellconfluency in the culture flask at less than 50%. Trypsin-EDTA (0.25%,Invitrogen) was used to detach the adherent cells from the flask duringpassage. Typical expansion of the “slowly-adhering cells” took anaverage of 17 days (starting from the day production is initiated) toachieve an average total viable cell number of 37 million cells.

Once the desired cell number was achieved, the cells were harvested fromthe flask using Trypsin-EDTA and centrifuged (2,500×g, 5 minutes). Thepellet was resuspended in BSS-P solution (HBSS supplemented with humanserum albumin (2% v/v, Sera Care Life)) and counted. The cell solutionwas then centrifuged again (2,500×g, 5 minutes), resuspended withCryopreservation Medium (CRYOSTOR™ (Biolife) supplemented with humanserum albumin (2% v/v, Sera Care Life Sciences)) to the desired cellconcentration, and packaged in the appropriate vial for cryogenicstorage. The cryovial was placed into a freezing container and placed inthe −80° C. freezer. Cells were administered by thawing the frozen cellsuspension at room temperature with an equal volume of physiologicsaline and injected directly (without additional manipulation). Thelineage characterization of the slowly adhering cell populations showed:Myogenic (87.4% CD56+, 89.2% desmin+), Endothelial (0.0% CD31+),Hematopoietic (0.3% CD45+), and Fibroblast (6.8% CD90+/CD56−).

Analysis for Characterization of Enriched and Isolated MDCs

Following disassociation of the skeletal muscle biopsy tissue, twofractions of cells were collected based on their rapid or slow adhesionto the culture flasks, as described above. The cells were then expandedin culture with growth medium and then frozen in cryopreservation medium(3×10⁵ cells in 15 μl) in a 1.5 ml eppendorf tube, also as describedabove. For the control group, 15 μl of cryopreservation medium alone wasplaced into the tube. These tubes were stored at −80° C. untilinjection. Immediately prior to injection, a tube was removed fromstorage, thawed at room temperature, and resuspended with 15 μl of 0.9%sodium chloride solution. The resulting 30 μl solution was then drawninto a 0.5 cc insulin syringe with a 30 gauge needle. The investigatorperforming the surgery and injection was blinded to the contents of thetubes.

Cell count and viability was measured using a Guava flow cytometer andViacount assay kit (Guava). CD56 was measured by flow cytometry (Guava)using PE-conjugated anti-CD56 antibody (1:50, BD Pharmingen) andPE-conjugated isotype control monoclonal antibody (1:50, BD Pharmingen).Desmin was measured by flow cytometry (Guava) on paraformaldehyde-fixedcells (BD Pharmingen) using a monoclonal desmin antibody (1:100, Dako)and an isotype control monoclonal antibody (1:200, BD Pharmingen).Fluorescent labeling was performed using a Cy3-conjugated anti-mouse IgGantibody (1:250, Sigma). In between steps, the cells were washed withpermeabilization buffer (BD Pharmingen). For creatine kinase (CK) assay,1×10⁵ cells were plated per well into a 12 well plate indifferentiation-inducing medium. Four to 6 days later, the cells wereharvested by trypsinization and centrifuged into a pellet. The celllysis supernatant was assayed for CK activity using the CK LIQUI-UV® kit(Stanbio).

Example 3 Treatment of Urinary Incontinence with Human MDCs in a RatModel

Treatment with human MDCs led to restoration of leak point pressure(LPP) back to normal levels in an experimental model of stress urinaryincontinence (SUI). Injected human MDCs alleviated urinary incontinencein a well established rat model.

These experiments demonstrate proof of concept and feasibility of usinghuman cell therapy for urologic application. The human MDCs wereharvested from a clinically-obtainable sized muscle biopsy, and improvedphysiologic outcomes for up to four weeks in an immunocompromised ratmodel of SUI. Histologic evaluation demonstrated periurethral muscleatrophy in the sham group only. Human MDCs were present in the nude raturethral sphincter 4 weeks after injection. Treatment with human MDCsled to restoration of LPP back to near-normal levels in an experimentalmodel of SUI in the nude rat.

Animals:

The experiments described below were performed using 6-8 week oldfemale, athymic nude rats (Hsd:RH-mu, Harlan Laboratory). Proceduralprotocols were approved by the Animal Research Care Committee ofChildren's Hospital of Pittsburgh. The policies and procedures of theanimal laboratory are in accordance with those detailed in the guide forthe ‘Care and Use of Laboratory Animals’ published by the US Departmentof Health and Human Services.

Denervation of Sciatic Nerve (SUI Model):

A well-established SUI model was created through bilateral transectionof the sciatic nerve. Rats were given isoflurane anesthesia (2 L/min)and, after appropriate induction, bilateral vertical dorsal incisionswere performed over the ischiorectal fossa. Under an operatingmicroscope, the sciatic nerve on each side was identified and 2 mm ofthose trunks were excised distal to its origin from the vertebralcolumn, but proximal to the branching of the pudendal nerve.

Human MDC Isolation:

Human MDCs used in this study were isolated from human skeletal muscletissue (˜250 mg) harvested from the rectus abdominus of a single donor,and isolated according to the single plate technique described above.Culture expansion was carried out in an antibiotic-free proprietarymedium supplemented with 10% fetal bovine serum. Flow cytometricanalysis of the MDC suspensions was performed to evaluate myogeniccontent through antibody labeling of CD56 expression (BD Pharmingen).MDCs were cryopreserved at a concentration of 1×10⁶ viable cells/10 μL.Separate aliquots of carrier medium alone were also prepared for shaminjection.

Injection Procedure:

Seven days following denervation, under isoflurane anesthesia (2 L/min),a low midline incision was made to expose the bladder and urethra.Cryopreserved MDCs or sham suspensions were thawed with an equal volumeof saline just prior to injection. A 3/10-mL insulin syringe was used toinject either 10 μL (5×10⁵ cells) of MDC suspension or sham aliquot intoeach lateral wall of the mid-urethra with microscopic guidance.Non-denervated, non-injected, age-matched animals served as controls.

In Vivo Cystometry (CMG) and Leak Point Pressure (LPP) Measurement:

In vivo functional measurements were performed 4 weeks followinginjections. Under urethane anesthesia (1.2 g/kg subcutaneous injection),a midline abdominal incision was made and the ureters were ligated. Atransvesical catheter with a fire-flared tip (PE-90) was inserted intothe dome of the bladder for bladder filling and pressure recording, andthe abdomen was closed. A three-way stopcock was connected to thetransvesical tube to monitor the bladder pressure during cystometry(continuous infusion of normal saline at rate of 0.04 mL/min). Thevoided volume, bladder capacity and maximal voiding pressure weremonitored. After cystometry, all rats underwent spinal cord transectionat the T9 level in order to eliminate spontaneous bladder activity inresponse to increasing intravesical pressures. The rats were thenmounted on a tilt table and placed in the vertical position.Intravesical pressure was clamped by connecting a large 50 mL syringe tothe bladder catheter and the pressure transducer via PE-190 tubing andthree-way stopcocks. The reservoir was mounted on a metered verticalpole for controlled height adjustment. Intravesical pressure wasincreased in 1-3 cmH₂O steps from zero upward until visualidentification of leakage; this pressure was identified as the LPP.Three consecutive readings were obtained and averaged for each animaland presented as a single LPP.

Tissue Harvest and Histology:

Immediately following the LPP measurement, the entire urethra-bladdercomplex was removed. The tissues were snap frozen using 2-methylbutaneprecooled in liquid nitrogen. Cryosections of the urethra were labeledwith hematoxylin/eosin (H&E) for general histology, and alsoimmunofluorescently-labeled with human specific anti-lamins A/C antibody(Novocastra, U.K.) to follow the fate of the injected MDC.

Statistical Analysis:

Data are presented as means±SE. Overall comparisons between groups wereperformed using a one-way analysis of variance (Tukey's multiplecomparison test). A p-value of less than 0.05 was accepted assignificant.

The injected MDC suspensions contained 87.7% myogenic (CD56-positive)cells; the remainders of the cells were fibroblastic. There were noserious adverse effects observed in any rat in the control, sham andMDC-injected groups. However, partial obstruction of the externalurethral meatus due to infection at the perineal area was found in oneanimal each in both the sham and human MDC-injected groups. Thus, theseanimals were excluded from the functional analysis.

CMG and LPP Measurement:

No difference in any measured cystometric parameter was observed betweenthe control, sham and human MDC-injected groups (Table 2).

TABLE 2 Cystometric variables in each group. Cystometric parametersControl Sham MDC P-value Maximal voiding 29.8 ± 1.4  29.3 ± 2.8  33.7 ±5.8  0.677 pressure (cmH₂O) Bladder capacity 0.40 ± 0.06 0.36 ± 0.090.34 ± 0.03 0.827 (ml)

Denervation of the urethral sphincter resulted in a significant decreasein LPP from the control to sham groups (FIG. 1) (43.4±0.6 to 27.8±0.7cmH₂O, respectively; p<0.05). LPP was restored to significantly higherlevels following MDC injection (35.7±2.0 cmH₂O) when compared to thesham group (p<0.05); however, at the 4 week time point, this level ofrestoration remained significantly less than that of control group(p<0.05).

Histological Analysis:

In the denervated rats, the proximal urethral sphincter was atrophic at4 weeks compared with control (FIG. 2). Human nuclei present within therat sphincter tissue was revealed through immunofluorescent labelingusing a human-specific antibody to the nuclear envelope proteins laminsA and C. Tissues from the MDC-injected group showed clear positivelabeling of numerous human nuclei incorporated within the external(striated) sphincter muscle (FIG. 3).

All patent applications, patents, texts, and literature references citedin this specification are hereby incorporated herein by reference intheir entirety to more fully describe the state of the art to which thepresent invention pertains.

As various changes can be made in the above methods and compositionswithout departing from the scope and spirit of the invention asdescribed, it is intended that all subject matter contained in the abovedescription, shown in the accompanying drawings, or defined in theappended claims be interpreted as illustrative, and not in a limitingsense.

We claim:
 1. A method for preparing a cell population containing musclederived cells (MDCs) useful for administration to treat urinary tractdisease in a mammalian subject, comprising: (a) suspending cellsisolated from human skeletal muscle in a first cell culture containerfor a duration sufficient to adhere a first cell population to thecontainer and to leave a second cell population remaining unadhered andin a culture medium in the container; (b) transferring the culturemedium and second cell population from the first cell culture containerto a second cell culture container; (c) allowing cells from the secondcell population to attach to the second cell culture container; and (d)isolating the cells attached to the second cell culture container toobtain said cell population containing MDCs.
 2. A method for preparing acell population containing muscle derived progenitor cells (MDCs),comprising: mincing skeletal muscle tissue obtained from a human patientto be treated with muscle derived progenitor cells, wherein the skeletalmuscle tissue has been stored under storage conditions causing stress tothe skeletal muscle tissue; digesting the minced muscle tissue to obtaina mixed population of cells; and preparing an enriched population ofmuscle derived progenitor cells from the mixed population of cells. 3.The method of claim 2, wherein said storage conditions include cooling.4. The method of claim 2, wherein said storage conditions includestoring the skeletal muscle tissue in hypothermic medium.
 5. The methodof claim 4, wherein the hypothermic medium contains an antibiotic. 6.The method of claim 4, wherein said storage conditions include storingthe skeletal muscle tissue at a temperature from about 4° C. to roomtemperature.
 7. The method of claim 2, wherein the enriched populationof muscle derived progenitor cells is prepared based on differentialadherence characteristics of cells of the mixture of cells.
 8. Themethod of claim 7, wherein said preparing an enriched population ofmuscle derived progenitor cells includes suspending said mixture ofcells in a first cell culture container for a period sufficient for afirst population of cells to adhere to the container and a secondpopulation of cells to remain non-adhered to the container, andtransferring the second population of cells to a second cell culturecontainer.
 9. The method of claim 2, also comprising freezing theenriched population of muscle derived progenitor cells in the presenceof cryopreservation medium.
 10. The method of claim 2, wherein theskeletal muscle tissue has been stored for 1 to 7 days under storageconditions causing stress to the skeletal muscle tissue
 11. A method oftreating a urinary tract disease in a human patient in need thereof,comprising administering to the urinary tract of the patient an enrichedpopulation of muscle derived progenitor cells prepared in accordancewith claim 2.